Gene targeting (GT) is a technique of modifying a target DNA on a genome at will by recombination utilizing the base sequence homology of DNAs. In the field of plants also, this technique is very promising in the fundamental research and in the development of breeding materials.
Nevertheless, the frequency of the homologous recombination in higher plants is low. When vector shaving a certain mutation on a sequence homologous to a target DNA (GT vectors) are introduced into cells from the outside to modify the target DNA via GT, most of the vectors are randomly inserted into the genomes. Against this background, positive-negative selection has been developed to efficiently select cells in which GT has successfully occurred. This method is a selection method in which cells having GT vectors randomly incorporated in the genomes are eliminated on the basis of the expression of a negative selectable marker gene, while cells having mutations introduced in target DNAs by GT are isolated on the basis of the expression of a positive selectable marker gene.
However, when this method is used, the expression cassette of the positive selectable marker gene remains in the target DNA. Accordingly, this cassette needs to be removed in a case where only a required mutation is to be introduced into a target DNA. In this regard, there has been a report so far on a system in which a positive selectable marker gene is removed after GT using a site-specific recombinase. Nevertheless, when this system is used, the recognition sequence of the site-specific recombinase remains after the marker is removed. Since it is also reported that even inserting a short base sequence influences the expressions of adjacent genes, there has been a demand for the development of a technique capable of marker removal without leaving any footprint after GT, and used when an introduction system is constructed for a mutation equivalent to a spontaneous mutation.
Regarding such a technique, there has been a report that only a required mutation was successfully introduced into a target DNA in mammalian cells by utilizing a special transposon, piggyBac transposon (hereinafter also referred to as “piggyBac”), which leaves no footprint after the transposition (NPL 1). To be more specific, it has been revealed that, after the modification of target DNAs by GT, transiently expressing a piggyBac transposase (hereinafter also referred to as “transposase”) in mouse- and human-derived culture cells made is possible to remove positive selectable marker genes from the target DNAs.
Nevertheless, in such a mutation introduction system for mammalian cells, the efficiency of removing piggyBac and positive selectable marker genes is so low that a negative selection needs to be performed to obtain cells from which piggyBac and positive selectable marker genes are removed. Further, in cells selected in this manner, piggyBac is re-inserted in a different genome region from a target DNA at a frequency as high as 68 to 79%. Moreover, since the piggyBac is an insect-derived transposon, whether the transposition is possible in plant cells as in the cases of mammalian cells has not been revealed.
Accordingly, in order to verify whether unnecessary sequences can be removed by utilizing the piggyBac transposition in plant cells also, the present inventors have constructed a system in which a vector having a reporter gene incorporated in piggyBac is randomly inserted in the genomic DNAs of plant cells. Then, the result of constitutively expressing the transposase in this system has revealed that the inserted reporter gene can be removed together with the piggyBac without leaving a trace (NPLs 2 to 5).
However, in this system, although the efficiency of removing piggyBac randomly inserted in the genomic DNAs of the plant cells is approximately 72%, the percentage of the piggyBac removed but inserted again into the genomic DNAs is as high as 41%. Thus, when this system is utilized to introduce only a required mutation into a target DNA of plant cells, further operations are required to remove cells in which piggyBac is re-inserted in regions other than the target DNA.